M sub(3) muscarinic receptors on murine HSDM1C1 cells: Further functional, regulatory, and receptor binding studies

In the present studies, the pharmacology and regulation of the functional muscarinic receptors on HSDM1C1 cells were probed using phosphoinositide (PI) turnover assays. In addition, the receptor binding of the putative M sub(3)-selective radioligand, [ super(3)H]4-DAMP, to cell homogenates was characterized. Carbachol, (+)muscarine and cis-dioxolane were full agonists which stimulated PI turnover by 13.3 plus or minus 1.0 fold above basal values. The potencies of numerous agonists in this assay system were relatively similar to their affinities in receptor binding assays. Exposure of HSDM1C1 cells to 10 nM-10 mu M muscarine during the last 24h of [ super(3)H]myo-inositol-labeling resulted in a concentration-dependent reduction in the cis-dioxolane affinity and maximal PI response induced by subsequent treatment with cis-dioxolane. Pertussis toxin caused a partial reduction in the cis-dioxolane-induced PI turnover. Likewise, exposure of the HSDM1C1 cells to an active phorbol ester (TPA) resulted in a partial inhibition of the cis-dioxolane-induced PI turnover. [ super(3)H]4-DAMP binding to cell homogenates was of high affinity and moderate capacity. The pharmacological specificity (4-DAMP > p-FHHSiD > dicyclomine > pirenzepine > methoctramine > AFDX-116 > gallamine) resembled that for [ super(3)H]NMS binding and correlated well with that observed for inhibition of PI turnover. The studies further support the identification of M sub(3) receptors on HSDM1C1 cells. These receptors have been shown to be influenced by pertussis toxin, an active phorbol ester and to exhibit desensitization.